Introduction

Angiodysplasia of the skin is abnormally dilated blood vessels, including capillaries, located under the basal growth zone of the epidermis. In most cases, 60–80%, they are localized in the face, neck, and upper body, less often on the extremities. Angiodysplasia can have various forms of manifestations: diffuse, linear, stellate, and others, and also differ in the type of blood filling (arterial, mixed, venous). This vascular pathology can be of congenital and acquired origin. In newborns, capillary angiodysplasia of the skin occurs in 0.3–0.5% of cases, and in older children, including acquired forms, on average more than 2.6% [1,2,3,4,5,6]. Due to the fact that this vascular pathology can reduce the quality of life of a child and his entire family, timely and effective treatment is a significant social and medical problem [4, 6].

To date, all existing methods of treatment are ineffective, have a number of disadvantages, and do not provide the necessary clinical and aesthetic results of treatment [1,2,3,4,5,6,7,8]. The general therapeutic effect often leads to the development of negative consequences of a somatic nature on the part of the cardiac and pulmonary systems, neurological disorders, and others [4, 6, 9]. Local methods of treatment often require multiple manipulations or sessions and can cause irreversible damage to the skin, up to cicatricial deformity [1, 4, 9,10,11].

Currently, photodestruction by laser radiation is considered the most effective and promising method of treating skin angiodysplasia. However, with the proposed methods with non-selective laser radiation, uncontrolled thermal damage to all tissue structures of the skin with the subsequent development of cicatricial deformation of the skin, hyperpigmentation, and other undesirable consequences in the affected area is not excluded [1, 2, 4, 9,10,11,12,13,14,15,16].

In this regard, the problem of treating this pathology remains relevant, which determines the need to create a new method of photodestruction based on the principle of selectivity of action on pathological subepithelial vascular structures of the skin with minimal damage to other epithelial tissues.

This can only provide the correct choice of laser radiation with a wavelength that is predominantly absorbed by chromophores by hemoglobin and oxyhemoglobin in the vascular structures of biological tissue, their further hardening, and regression with a minimal traumatic component of healthy skin structures, including the growth zone [4, 9, 11,12,13,14,15,16].

In the spectral range, laser radiation with a wavelength of 520 ± 5 nm is located near the absorption peaks for hemoglobin and oxyhemoglobin [13]. In this regard, it can be considered the most promising for influencing subepithelial vascular structures, including capillary angiodysplasia, with different blood patterns.

An earlier experimental study in vitro on model biological objects (cooled samples of the skin and liver of minipigs (Sus salvanius) of the Svetlogorsk population) confirmed the selectivity of absorption of “green” laser radiation with a wavelength of 520 ± 5 nm by liver tissues containing a much larger amount of hemoglobin compared to the skin [17, 18]. The results of this in vitro study determined the possibility of using “green” laser radiation with a wavelength of 520 ± 5 nm for the purposes of selective photothermolysis of vascular structures, including subepithelial angiodysplasia, which was confirmed in this work.

Biomedical experimental study in vivo

An experimental study in vivo on the combs of live white chickens was carried out to clarify the nature of selective photothermolysis and to study the regenerative wound process in the zone of exposure to “green” laser radiation with a wavelength of 520 ± 5 nm. The results of the study determined the prospect of using this radiation for the selective photodestruction of subepithelial pathological vascular structures, in particular, angiodysplasia of the skin.

Experimental equipment, materials, and methods

Equipment and modes of laser radiation

We used an experimental sample of a solid-state laser apparatus based on semiconductor diodes operating at a radiation wavelength of 520 ± 5 nm in the green spectral range, near the absorption peak of hemoglobin and oxyhemoglobin (Table 1).

Table 1 Technical and operational characteristics of the device
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The experimental sample was developed for the purposes of medicine at the Prokhorov General Physics Institute of the Russian Academy of Sciences [19, 20]. The prototype manufactured at the Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences (Fig. 1).

Fig. 1
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Medical laser device used for selective photodestruction

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In the experiment, we used “green” laser radiation with constant parameters: wavelength 520 ± 5 nm, average power 3 W, pulse repetition rate 10 Hz, laser spot diameter 0.8 mm. In the study, the following parameters of laser radiation were changed: pulse duration 2 ms, 5 ms, and 10 ms, exposure 5 s and 10 s. The study was carried out in four modes with different combinations of these parameters. The parameters of the modes used in the experimental study are presented in Table 2.

Table 2 Parameters of modes used in the experiment
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In this work, the surface of the object was affected in two ways, pointwise with a fixed spot and with the movement of the spot by manual scanning. In this case, the corresponding identical parameters of laser radiation were used.

Object of experimental research

The object was the red combs of biological models of live white Cross Hisex White chickens. The reason for choosing them as the closest model of subepithelial vascular formations was the presence of a large number of dilated capillaries in the subcutaneous vascular network and the absence of melanin pigment in the tissues. On the micrograph of the histo-preparation of the combs, dilated capillaries in the subepithelial zone are determined (Fig. 2).

Fig. 2
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Micrograph of a histological specimen of unchanged comb tissue

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The micrograph of unchanged comb tissue shows well-defined cells of stratified squamous epithelium; in the underlying stroma, there are a large number of congested capillaries with an expanded lumen, staining with hematoxylin and eosin, magnification × 200.

Experimental research methodology

The experiment involved 42 individuals of chickens with red combs, aged 7 to 8 months, each of which was marked with a ring on its paw.

The experiment with biological models was performed using general anesthesia by intramuscular administration of Zoletil, diluted in accordance with the instructions at the rate of 0.01 ml per 100 mg of live bird weight.

On the combs of the models, zones of exposure to “green” laser radiation with a wavelength of 520 ± 5 nm were formed, with the indicated mode parameters. Spot and scanned zones were applied only on one side of the ridge, using two modes of radiation parameters for each ridge, namely 1 and 2 for one ridge and on the other 3 and 4 modes. First, for each mode, 3 point zones were formed on the ridge, located for the mode with a lower intensity closer to the beak, for the second with more aggressiveness closer to the back of the head. Then, under them, at a distance of 1–2 mm, a strip of 1.0–2.0 mm wide, up to 10.0 mm long was applied by scanning at a speed of about 1 cm per second, using the corresponding point energy mode. For each pair of methods of exposure used, 3 biological models were used, that is, three chicken combs. A schematic procedure for the formation of zones of laser action on the combs of biological models is shown in Fig. 3.

Fig. 3
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The procedure for the formation of zones of laser exposure

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The sampling of the experimental material was carried out immediately after exposure—zero period, then on the 4th day, 7th, 12th, 21st, and 30th days, as well as after 3 months. The combs were harvested under general anesthesia with Zoletil by surgical excision with stitching on the surgical wound.

In the experiment, the determined changes in the zones of influence on live combs and after their cutting off on macro-preparations were studied, from which samples were prepared for subsequent histological examination. During the entire observation, the state of the tissues and the peculiarities of the course of the regenerative wound process, as well as the selectivity to hemoglobin-containing vascular structures in the affected zones, were investigated.

Methodology for performing histological examination

Tissue samples with laser exposure zones for histological examination were fixed in 10% neutral formalin. Further processing was carried out automatically in an Excelsior apparatus for histological wiring (TermoScientific, Great Britain) and embedded in paraffin using an EC350 apparatus (Microm, Germany). From paraffin blocks, sections with a thickness of 2–3 µm were obtained using an HM355S microtome with an STS section transfer system (Termo Scientific, Great Britain). The resulting histological preparations were dewaxed in successive changes of xylene and alcohol solutions, and then stained with hematoxylin and eosin. The finished stained histological preparations were placed under a cover glass and examined under a CX41 microscope (Olympus, Japan).

A PannoramicMidi scanning microscope (3DHISTECH Hungary) was used to obtain high-precision images of histological preparations. Software processing of the images of histological preparations made it possible to assess their morphometric parameters with the measurement of areas of damaged tissues in the zones of laser exposure.

Research results

Examples of the results of a morphological macroscopic study of the zones of exposure to laser radiation with a wavelength of 520 ± 5 nm at various times after exposure to laser radiation with parameters of modes 1 to 4 on chicken combs samples are shown in Fig. 4(a–j).

Fig. 4
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a 0 term, mode 1–2. The zones of exposure when using modes 1 and 2 with lower energy in spot application and in scanning mode are represented by whitish areas of even skin. b 0 term, mode 3–4. Point impact zones with higher energy at 3 and 4 modes have the form of small brownish craters no more than 1.5 mm in diameter, with a narrow yellowish rim up to 1.0 mm. In the case of scanning at mode 3, areas of white-yellow color are determined; at mode 4, there are light brown crusts in places, and the surface of the areas is even. c 12 days, mode 1–2. All affected areas have a whitish, flat surface of the skin; there are no signs of inflammation in the surrounding tissues. d 12 days, mode 3–4. The areas of influence are white with a flat slightly reduced surface; there are no inflammatory manifestations in the surrounding tissues (single deep defects are the result of damage by another individual). e 30 days, mode 1–2. In all areas of exposure, a relatively flat, whitish surface remains; deformation of the skin and inflammatory manifestations are not detected. f, g 30 days, mode 3–4. Comb and comb macro preparation. f All areas of exposure retain a whitish color with a flat surface; there is no deformation of the skin and no inflammatory changes. g These changes are preserved and are better defined on a macro-preparation of a comb; the entire surface of which becomes even. h 90 days, mode 1–2. In the affected areas, a persistent effect of white color and surface leveling is noted; there is no deformation of the skin and no inflammatory manifestations. i, j 90 days, mode 3–4. Comb and scomb macro-preparation. i In all areas of exposure, the effect of white color remains and the surface of the skin is relatively flat with a slight retraction; there are no deforming changes and inflammatory manifestations. j On the macro-preparation of the comb in the affected zones, white areas with a flat surface are clearly defined

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Based on the morphological macroscopic examination of the zones of exposure to laser radiation with a wavelength of 520 ± 5 nm, characteristic features were determined.

Immediately after the application of the zones of laser action, for all the parameters of the radiation regimes used, changes of a similar nature were noted, the severity of which somewhat increased with an increase in energy activity. With point application, the formation of shallow defects—craters—was observed and a relatively rapid recovery by 12 days in the form of whitish dots of areas of damaged skin.

During scanning, the surface of the skin immediately acquired a whitish color and became smoother in comparison with the surrounding skin, which persisted throughout the observation period, including 30 and 90 control days.

Thus, in all cases, regardless of the regime used and the method of applying laser radiation, by day 12, restoration of the skin surface with a whitish color in the affected areas was noted, and at all indicated periods, perifocal inflammatory changes in the tissues of the combs were not noted.

Examples of micrographs of histological preparations with laser scanning zones with the most energetically aggressive parameters of 4 modes are shown in Fig. 5(a–h).

Fig. 5
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a, b Micrograph of a histological specimen of a comb, 0 term, and its fragment with an area of unchanged comb tissue. a In the affected area, the preservation of horny masses on the surface and soft coagulation of stratified squamous epithelium, in the form of erasing the boundaries of cells with the absence of nuclei, while maintaining the basement membrane, is noted. In the underlying stroma, the closure of the lumens of the capillaries, the fusion, and homogenization of collagen fibers are noted. The depth of the determined changes is about 350 microns. Staining with hematoxylin and eosin. Magnification × 100. b Unchanged comb tissue with well-defined cells of stratified squamous epithelium and a large number of full-blooded capillaries with an expanded lumen in the underlying stroma. Staining with hematoxylin and eosin. Magnification × 100. c Micrograph of a histological specimen of a comb, day 7. A small depression on the surface is made with necrotic masses from the stratum corneum. The basal cellular structures of the epithelium are preserved; differentiated cellular structures of the epithelium are determined above them. In the subepithelial sections, collapsed capillary lumens remain; there is a slight edema of the stroma. Staining with hematoxylin and eosin. Magnification × 100. d, e Micrograph of a histological specimen of a comb, 21 days and its fragment with an area of unchanged comb tissue. d The epithelium with pronounced hyperkeratosis, its basal and cellular structures are differentiated. In the subepithelial sections, the collapsed lumens of the capillaries are preserved, and there is also a zone with a compact arrangement of large collagen fibers, followed by a zone of loose connective tissue with edema and thin collagen fibers. Staining with hematoxylin and eosin. Magnification × 200. e For comparison, unaltered comb tissue with stratified squamous epithelium and a large number of full-blooded capillaries with a wide lumen in the subepithelial zone. Staining with hematoxylin and eosin. Magnification × 200. e, f Micrograph of a comb preparation, 30 days. Slight hyperkeratosis of the epithelium, distinct cellular differentiation of the epithelium, including the basal layer. In the subepithelial regions, the lumens of the capillaries collapsed. Under the epithelium, there is a zone with a compact arrangement of large collagen fibers, and behind it is an area of loose connective tissue with slight edema and then a layer of thinner collagen fibers. Changes are noted down to a depth of 390 microns. Staining with hematoxylin and eosin. Magnification × 100. g, h Micrograph of a comb preparation, day 90 and its fragment with an area of unchanged comb tissue. g The epithelium with typical hyperkeratosis and its cellular structures, including the basal ones, are clearly defined. In the subepithelial regions, the lumens of almost all capillaries collapsed. In the subepithelial zone, there is a relatively wide and structurally pronounced collagenization zone with a compact arrangement of large collagen fibers parallel to the epithelium. This zone is followed by an area of loose connective tissue with thin, chaotically directed collagen fibers. Staining with hematoxylin and eosin. Magnification × 100. h For comparison, an area of unchanged comb tissue with a large number of full-blooded capillaries with a wide lumen in the stroma, underlying stratified squamous epithelium. Staining with hematoxylin and eosin. Magnification × 100

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Based on the analysis of the results of histological examination, characteristic changes and features of the tissue regeneration process after exposure to laser radiation with a wavelength of 520 ± 5 nm with the most energetically aggressive parameters of 4 modes were noted. In all cases, minimal damage was noted to the basal cellular structures of the epithelial growth zone, which, while maintaining their viability, provide the possibility of central epithelialization from day 7 to complete restoration of the differentiation of epithelial cell structures by day 21.

In the subepithelial zone, by the 21st day, the formation of a section of compactly located large collagen fibers is determined, the organization of which significantly increases by the 30th and 90th days, and in the subepithelial zone, the effect of closure of full-blooded capillaries with a wide lumen is preserved. At all designated periods and with all energy parameters of exposure, the epithelium is minimally damaged, while the depth of the zone of thermal damage to subepithelial tissue structures is from 350 to 390 μm with a relatively clear border with the underlying, unchanged tissues.

The severity of thermal destructive changes in the tissues of chicken comb samples at the same radiation power, 3 W, increases with an increase in energy activity, pulse duration, and exposure to pulsed exposure, while the nature of thermal damage to tissue structures of the combs continues to remain the same.

The discussion of the results

The peculiarity of the effect of laser radiation with a wavelength of 520 ± 5 nm lies in the predominant damage to the subepithelial vascular structures of the combs with minimal reversible damage to the epithelium, including the zone of its growth. This ensures persistent whitening and leveling of the skin surface in the affected areas, due to the closure of the full-blooded vessels with a wide lumen in the subepithelial zone, in the future the formation of a parallel organized surface of the collagenosis layer there, as well as the preservation of the epithelial growth zone and epithelialization of the wound surface in physiological terms, without the formation of cicatricial deformity of the skin.

This effect of exposure is based on the selective absorption of the used “green” laser radiation with a wavelength of 520 ± 5 nm, mainly by blood chromophores (hemoglobin, oxyhemoglobin) of subepithelial vessels, then their desolation and collagenization, as well as minimal absorption by other chromophores of skin tissues and preservation of its growth zone.

Thus, the results of the experimental study confirmed the selectivity of the action of laser radiation of the green spectrum with a wavelength of 520 ± 5 nm on the subepithelial vascular structures, which is one of the necessary conditions for precision photodestruction of angiodysplasia of the skin and some of its other vascular pathologies.

Conclusion

It has been experimentally substantiated that “green” laser radiation with a wavelength of 520 ± 5 nm ensures the selectivity of photodestruction of subepithelial vascular structures, with minimal reversible damage to the skin and without the formation of deforming scars. In the experiment, it was determined that for an effective effect on subepithelial vascular structures, it is sufficient to use a laser radiation power of 3 W, with a pulse duration range from 2 to 10 ms, with an exposure duration from 5 to 10 s, respectively, with a scanning speed of about 1.0 cm per second.

Selective photodestruction by laser radiation with a wavelength of 520 ± 5 nm, combining the precision of action on hemoglobin-containing subepithelial vascular structures with a minimal traumatic component of the epithelium, is promising for the treatment of various angiodysplasia of the skin, including capillary, and its use should ensure the achievement of the optimal possible clinical and aesthetic result, which needs to be confirmed by further clinical studies.